Human cytomegalovirus (HCMV) is a common infectious agent that can cause significant morbidity and mortality in immunosuppressed or immunocompromised individuals. HCMV is a major opportunistic pathogen in patients with AIDS as well as patients undergoing immunosuppressive therapy following organ transplantation. In addition, newborn infants that have been congenitally infected with HCMV develop severe complications such as mental retardation and hearing loss. Three characteristic phases of HCMV gene expression have been defined: immediate early (IE), early and late. The expression of viral early genes in HCMV infected cells is a complicated process involving the interaction of the viral IE proteins with cellular transcription factors. Genes expressed within the early phase of HCMV infection include those important for DNA replication. Thus once these genes are expressed, it is likely that the virus will proceed towards productive infection. Insights into early gene regulation are therefore crucial to our understanding of HCMV permissiveness, latency and reactivation. The overall objective of this proposal is to examine the regulation of early gene expression within the natural environment of the gene. In these studies, the regulation of viral early genes from the non-essential region of the unique short (US) component of HCMV will be assessed. Employing this non-essential region of the HCMV genome affords a unique opportunity to examine HCMV early gene regulation within the natural gene context. The role of both promoter and intragenic sequences on gene regulation will be examined within the viral genome. In addition, the influence of genomic environment on gene regulation will be assessed. In order to accomplish these goals, the following specific aims will be performed: 1) The influence of genome location on early gene expression will be determined. 2) Mapping of regulatory elements within US region early promoters using transient transactivation analysis. 3) Analysis of these potential regulatory elements within the natural genomic context. 4) The importance of sequences downstream of the promoter region in early gene regulation will be examined.